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Original Studies |
Arg972 Amino Acid Polymorphism in Insulin Receptor Substrate-1 Affects Glucose Metabolism in Skeletal Muscle Cells1
Laboratory of Molecular Medicine, Department of Internal Medicine, University of Rome-Tor Vergata (M.L.H., M.F., O.P., P.B., R.L., G.S.), Rome, Italy; Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health (D.L.), Bethesda, Maryland 20892; and Department of Medicine, Columbia University (D.A.), New York, New York 10027-6902
Address all correspondence and requests for reprints to: Giorgio Sesti, M.D., Dipartimento di Medicina Interna, Università di Roma-Tor Vergata, Via Orazio Raimondo, 00173 Rome, Italy. E-mail: sesti{at}uniroma2.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glucose transport is the rate-limiting step for glucose disposal in
skeletal muscle (25). Insulin stimulates glucose transport in skeletal
muscle through the translocation of glucose transporters to the plasma
membrane from an intracellular pool. Another important metabolic
response regulated by insulin in skeletal muscle is the activation of
glycogen synthesis through the activation of glycogen synthase.
Distinct experimental approaches have shown that PI 3-kinase plays an
important role in insulin-stimulated glucose transport and
translocation of glucose transporters. Studies in both cultured cell
lines and isolated muscle strip have shown that inhibition of PI
3-kinase activity by wortmannin or LY294002 inhibits both basal and
insulin-stimulated glucose transport by affecting GLUT1 and GLUT4
translocation to plasma membrane (26, 27). A variant of the p85
regulatory subunit of PI 3-kinase (
p85) lacking the binding site for
p110 catalytic subunit inhibits insulin-stimulated glucose uptake and
GLUT1 translocation to plasma membrane (28). Recently, the
serine/threonine kinase Akt was recognized as a downstream target of PI
3-kinase (29). Akt was found to mediate some metabolic responses
induced by insulin, including stimulation of glucose transport,
translocation of glucose transporters to the cell surface, inactivation
of glycogen synthase kinase-3 (GSK-3), and stimulation of glycogen
synthesis (30, 31, 32, 33, 34). Transfection studies in both 32D-IR cells have
shown that polymorphism at codon 972 of IRS-1 impairs PI 3-kinase
activity (35, 36). Although a defect in PI 3-kinase activity is
expected to affect downstream signaling involving the Akt/GSK-3
pathway, the only two studies that addressed the role of Akt in human
skeletal muscle from type 2 diabetic patients have led to divergent
results (37, 38). An vitro study showed a reduction in
insulin-stimulated Akt activity (37), whereas a more recent in
vivo study revealed normal Akt activity despite a decrease PI
3-kinase activity in response to insulin (38). Thus, in the skeletal
muscle, the impact of the Arg972-IRS-1 variant on
metabolic insulin signaling downstream of PI 3-kinase remains to be
elucidated. In this study we stably overexpressed both wild-type IRS-1
and Arg972-IRS-1 variant in L6 skeletal muscle
cells to investigate directly whether the Gly to Arg amino acid
substitution at codon 972 of IRS-1 affects Akt/GSK-3 activity, glucose
transport, expression and cell surface translocation of the glucose
transporters GLUT-1 and GLUT-4, and glycogen synthesis.
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Materials and Methods |
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Anti-IRS-1 and anti-rat PI 3-kinase antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). A polyclonal antibody to the IRS-2 protein was prepared by immunizing rabbits with a synthetic peptide corresponding to residues 1310–1322 of the IRS-2 sequence. Antiphosphotyrosine and anti-GSK-3 polyclonal antibodies were obtained from Transduction Laboratories, Inc. (Lexington, KY). Anti-Akt antibody that recognizes both Akt1 and Akt2 and antiphospho-Akt (Ser473) antibody were obtained from New England Biolabs, Inc. (Beverly, MA). Polyclonal antibodies to the COOH-terminal sequences of GLUT1 and GLUT4 were raised in rabbits according to previous described methods (39).
Construction and expression of human IRS-1 complementary DNAs (cDNAs) in L6 cells
pcDNA3 expression vectors containing human wild-type IRS-1 (pcDNA3-WT-IRS-1) or Arg972 variant (pcDNA3-Arg972-IRS-1) were constructed as previously described (40). The L6 rat skeletal muscle cells grown in DMEM containing 10% FCS were transfected with 1 µg pcDNA3 alone, wild-type pcDNA3-WT-IRS-1, or pcDNA3-Arg972-IRS-1 suspended with 20 µl Lipofectamine in 1 mL serum-free DMEM, according to the manufacturer’s instructions. The cells were incubated with a selective medium containing 0.4 mg/mL geneticin G-418 sulfate. Individual G-418-resistant stable cell clones were cultured in DMEM containing 10% FCS and maintained in continuous passages (<15) by trypsinization of subconfluent cultures. L6 myoblasts were differentiated into myotubes according to previously described method (41). All of the studies described in this work were performed with stably transfected L6 cell clones at the myotube stage, as monitored by quantitating the percentage of nuclei in myotubes using phase contrast microscopy and by assessing creatine kinase activity. L6 myotubes were washed three times and lysed for 45 min at 4 C in lysis buffer A containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.6), 1 mmol/L MgCl2, 1 mmol/L CaCl2, 2 mmol/L ethylenediamine tetraacetate, 10% glycerol, 10 mmol/L Na4P2O4, 100 µmol/L sodium orthovanadate, 10 mmol/L NaF, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 2 mmol/L phenylmethylsulfonylfluoride, and 1.5% Nonidet P-40. Insoluble material was removed by centrifugation, and the supernatant was saved for analysis by Western blotting. The protein content of each sample was determined using the Bradford assay.
Tyrosine phosphorylation of IRS-1 and association of IRS-1 or IRS-2 with the p85 subunit of PI 3-kinase
L6 myotubes cultured in 100-mm dishes were incubated overnight in serum-free DMEM containing 0.25% BSA and subsequently incubated in the same buffer in the presence or absence of 100 nmol/L insulin for 2 min at 37 C. The cells were then washed three times and lysed for 45 min at 4 C in lysis buffer A. Insoluble material was removed by centrifugation, and the supernatant was incubated for 16 h at 4 C with either anti-IRS-1 or anti-IRS-2 antibody. Immune complexes were collected by incubation with protein A-Sepharose for 2 h at 4 C and resuspended in Laemmli buffer. Equal amounts of cell lysates or immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions. Proteins resolved by SDS-PAGE were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Aliquots of cell lysates for quantitating IRS-1 or IRS-2 cellular content were blotted with anti-IRS-1 or anti-IRS-2 antibody. Aliquots of IRS-1 or IRS-2 immunoprecipitates for quantitating IRS phosphorylation were blotted with antiphosphotyrosine antibody. Aliquots of IRS-1 or IRS-2 immunoprecipitates for measuring IRS-1 or IRS-2 binding to the p85 subunit of PI 3-kinase were blotted with anti-PI 3-kinase antibody. After extensive washings, the blots were incubated with peroxidase-conjugated goat antirabbit IgG antibodies. Proteins were detected using enhanced chemiluminescence, and band densities were quantified by densitometry.
IRS-1-associated PI 3-kinase activity
L6 myotubes expressing either WT-IRS-1 or Arg972-IRS-1 were treated with insulin for 2 min and then lysed. Cellular extracts were subjected to immunoprecipitation with anti-IRS-1 antibody, the immunoprecipitates were washed, and PI 3-kinase activity was assayed in the immunoprecipitates according to a previously described method (35, 36).
Akt phosphorylation and kinase activity
L6 myotubes expressing either WT-IRS-1 or
Arg972-IRS-1 were incubated for 2 h in
serum-free DMEM containing 0.25% BSA. The cells were then incubated in
the same buffer in the presence or absence of 100 nmol/L insulin for 2
min at 37 C. The cells were washed three times with PBS and lysed in
lysis buffer B containing 20 mmol/L Tris (pH 7.5), 1 mmol/L
ethylenediamine tetraacetate, 1 mmol/L
ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 1% Triton X-100, 5 mmol/L
Na4P2O4,
10 mmol/L ß-glycerolphosphate, 1 mmol/L sodium orthovanadate, 1
µg/mL leupeptin, and 0.1 mmol/L phenylmethylsulfonylfluoride. Cell
lysates were incubated with anti-Akt-
antibody, and immunocomplexes
were collected by incubation with protein G-Sepharose.
Immunoprecipitated proteins were washed and resuspended in kinase
buffer containing 20 mmol/L Tris-HCl (pH 7.5), 75 mmol/L
MgCl2, and 1 mmol/L dithiothreitol to which 500
µmol/L ATP, 10 µCi [
-32P]ATP, and 1 µg
GSK-3 peptide (GRPRTSSFAEG) had been added. After 20 min at 30 C,
aliquots were spotted on squares of P81 phosphocellulose paper, washed,
and counted using the Cherenkov method (32). In another set of
experiments, cell lysates were immunoprecipitated with anti-Akt
antibody, and immunocomplexes were subjected to SDS-PAGE, transferred
to PVDF membranes, and immunoblotted with either anti-Akt or
antiphospho-Akt (Ser473) antibody.
GSK-3 activity
L6 myotubes expressing either WT-IRS-1 or
Arg972-IRS-1 were cultured for 2 days in DMEM
containing 0.5% FCS and incubated for 2 h in serum-free DMEM
containing 0.25% BSA. The cells were then incubated in the same
buffer in the presence or absence of 100 nmol/L insulin for 2 min at 37
C, washed three times, and lysed in lysis buffer B. Cell lysates were
immunoprecipitated with anti-GSK-3 antibody, washed, and resuspended in
25 mmol/L ß-glycerolphosphate, 40 mmol/L HEPES (pH 7.2), 10 mmol/L
MgCl2, and 2 mmol/L protein kinase inhibitor to
which 100 µmol/L ATP, 3 [-32P]ATP, and 1
µg phosphoglycogen synthase peptide had been added. After 20 min at
37 C, the reaction was stopped, and aliquots were spotted on squares of
P81 phosphocellulose paper and counted using the Cherenkov method.
Glucose transport and glycogen synthesis
2-Deoxy-D-[14C]glucose uptake was determined as previously described (41). For glycogen synthesis studies, L6 myotubes were incubated with DMEM/1% BSA containing 25 mmol/L HEPES (pH 7.6), and glucose (2.5 mmol/L, final concentration) for 3 h at 37 C. Insulin at the indicated concentrations and [U-14C]glucose (4 µCi) were then added to each well, followed by a 2-h incubation. Wells were washed with PBS, and cells were solubilized in 0.5 mL 30% KOH containing 2 mg unlabeled glycogen and incubated for 30 min at 37 C. The mixture was boiled for 15 min, and glycogen was precipitated in 70% ethanol on ice. The precipitate was pelleted by centrifugation, washed with 70% ethanol, and dissolved in water. Radioactivity was determined by scintillation counting. The glycogen synthase assay was carried out as previously described (41).
Preparation of plasma membranes and detection of the glucose transporters GLUT1 and GLUT4
Preparation of plasma membranes from L6 myotubes was carried out as previously described (41). For determination of total transporter content, cells were lysed in lysis buffer, and insoluble material was removed by centrifugation. Both plasma membrane fraction and total cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with either anti-GLUT1 or anti-GLUT4 antibodies.
Statistics
Differences between the cells lines were tested by unpaired Student’s t test. When appropriate, a two-way ANOVA test was used to compare data from the cell lines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L6 myoblasts were stably transfected with expression vectors for
human WT-IRS-1 or the Arg972-IRS-1 variant. In
all transfected cell clones used in this study, expression of both
WT-IRS-1 and Arg972-IRS-1 was 2.5- to 3-fold
higher than that of the endogenous IRS-1, as detected by immunoblotting
(Fig. 1
, top). Similar amounts
of recombinant IRS-1 were observed in cells expressing the WT-IRS-1 or
Arg972-IRS-1 (Fig. 1, top). L6
myoblasts stably transfected with the WT-IRS-1 (L6-WT) or
Arg972-IRS-1 (L6-Arg972)
maintained the ability to differentiate into myotubes. Two independent
clones of L6 cells expressing wild-type IRS-1 and two independent
clones of L6 cells expressing variant IRS-1 were used for further
experiments. Expression of recombinant IRS-1 did not change in L6-WT or
L6-Arg972 cells maintained in continuous passages
(<15), as monitored by immunoblotting.
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We first determined whether the Gly
Arg972
substitution altered tyrosine phosphorylation of IRS-1 and its ability
to activate PI 3-kinase. The extents of basal and insulin-stimulated
tyrosine phosphorylation of IRS-1 were similar in both L6-WT and
L6-Arg972 (Fig. 1
, middle). In
unstimulated cells, basal binding of p85 subunit to IRS-1 was 2.77- and
2.31-fold higher in L6-WT and L6-Arg972 cells,
respectively, compared with that in control L6 cells. Upon insulin
stimulation, binding of p85 subunit to IRS-1 was increased by 2.24-,
1.80-, and 1.81-fold in L6-WT, L6-Arg972, and
control L6 cells, respectively (Fig. 1, bottom). Thus,
compared with L6-WT cells, L6-Arg972 cells showed
a 17% decrease in binding of p85 subunit to IRS-1 under basal
conditions (P < 0.01) and a 20% decrease in response
to insulin (P < 0.001; Fig. 1, bottom).
Consistent with these results, basal PI 3-kinase activity associated
with IRS-1 was 2.0- and 1.64-fold higher in L6-WT and
Arg972-IRS-1 cells, respectively, compared with
that in control L6 cells (Fig. 2). Upon
insulin stimulation, PI 3-kinase activity associated with IRS-1 was
increased by 8.5-, 6.8-, and 5-fold in L6-WT,
L6-Arg972, and control L6 cells, respectively
(Fig. 2). Compared with L6-WT, L6-Arg972 cells
exhibited an 18% (P < 0.001) reduction of basal
IRS-1-associated PI 3-kinase activity and a 20% decrease in response
to insulin (P < 0.001; Fig. 2).
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Expression of endogenous IRS-2 did not change in L6 cells
expressing either wild-type or Arg972 variant
IRS-1 compared with that in control L6 cells (Fig. 3, top). Furthermore, the
extent of binding of p85 subunit to endogenous IRS-2 was similar in
both L6-WT and L6-Arg972 after insulin
stimulation (Fig. 3, bottom).
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In the basal state, the initial rate of
2-deoxy-D-glucose transport was 2.1- and 1.55-fold higher
in L6-WT and L6-Arg972 cells, respectively,
compared with that in control L6 cells (0.9, 1.87, and 1.40
nmol/mg·min for control L6, L6-WT, and
L6-Arg972, respectively; P <
0.0001). Thus, in L6 cells expressing the variant
Arg972-IRS-1, basal glucose transport was
decreased by 26% compared with that in L6 cells expressing wild-type
IRS-1 (P < 0.001). At submaximal insulin
concentrations, glucose transport was significantly increased in L6-WT
compared with L6-Arg972 and control L6 cells
(P < 0.001), whereas no differences were observed
between L6-Arg972 cells and control L6 cells
(Fig. 4). In response to
10-9 mol/L insulin, glucose transport was
increased by 120%, 70%, and 60% over basal in L6-WT,
L6-Arg972, and control L6 cells, respectively,
(P < 0.001; Fig. 4). The maximally insulin-stimulated
glucose transport was significantly increased in both L6-WT and
L6-Arg972 compared with control L6 cells. In
response to 10-7 mol/L insulin, glucose
transport was increased by 216%, 174%, and 152% over basal in L6-WT,
L6-Arg972, and control L6 cells, respectively.
Compared with L6-WT, L6-Arg972 cells exhibited a
42% decrease in submaximal and a 20% decrease in maximal
insulin-stimulated glucose transport (P < 0.001).
Using the data shown in Fig. 4, we calculated the insulin concentration
that results in 50% of the maximal insulin-stimulated glucose
transport (ED50). The ED50
was significantly higher (6.7 nmol/L) in
L6-Arg972 than in L6-WT cells (2.4 nmol/L;
P < 0.0001; Fig. 4, inset).
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There was no significantly difference in total GLUT1 and GLUT4
protein contents among control L6, L6-WT, and
L6-Arg972 cells (data not shown). Similar to
basal glucose transport activity, the amounts of GLUT1 and GLUT4 in
plasma membrane of unstimulated cells was significantly higher in both
L6-WT and L6-Arg972 cells compared with those in
control L6 cells (Fig. 5). In unstimulated L6-WT cells, the amounts of
GLUT1 and GLUT4 in plasma membrane were 3.2- and 3.6-fold higher than
those in control L6 cells. In unstimulated
Arg972-L6 cells, the amounts of GLUT1 and GLUT4
in plasma membrane were 2.66- and 2.65-fold higher than those in
control L6 cells. Compared with L6-WT, L6-Arg972
cells exhibited 17% and 27% decreases, respectively, in GLUT1 and
GLUT4 plasma membrane contents under basal conditions
(P < 0.01; Fig. 5). The
amount of GLUT1 translocated to the plasma membrane after insulin
stimulation was increased by 1.65-, 1.36-, and 1.21-fold in L6-WT,
L6-Arg972, and control L6 cells, respectively
(Fig. 5A). Similarly, the amount of GLUT4 translocated to the plasma
membrane in response to insulin was increased by 2.30-, 1.83-, and
1.81-fold in L6-WT, L6-Arg972, and control L6
cells, respectively (Fig. 5B). Compared with L6-WT,
L6-Arg972 cells exhibited an 18% reduction in
GLUT1 translocation (P < 0.001) and a 21% decrease
GLUT4 translocation (P < 0.005) in response to
insulin. The results suggest that defects in both GLUT1 and GLUT4
translocation may account for the impairment in glucose transport
observed in L6 cells expressing the Arg972-IRS-1
variant.
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Expression of Akt did not differ among control L6, L6-WT, and
L6-Arg972 cells, as detected by immunoblotting
(Fig. 6, top). In unstimulated
cells, basal Akt phosphorylation was 2.14- and 1.62-fold higher in
L6-WT and Arg972-IRS-1 cells, respectively,
compared with that in control L6 cells. However, in unstimulated L6
cells expressing the Arg972-IRS-1 variant, Akt
phosphorylation was decreased by 24% compared with that in L6-WT cells
(P < 0.01). Insulin-induced Akt phosphorylation was
increased by 1.4-, 1.1-, and 1.1-fold in L6-WT,
L6-Arg972, and control L6 cells, respectively
(Fig. 6, bottom). Compared with L6-WT,
L6-Arg972 cells exhibited a 22%
(P < 0.001) decrease in insulin-stimulated Akt
phosphorylation (P < 0.001; Fig. 6, bottom). Consistent with these results, insulin-stimulated
Akt kinase activity was decreased by 25% in
L6-Arg972 compared with L6-WT cells
(P < 0.001; data not shown).
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In unstimulated cells, basal GSK-3 activity was decreased by 56%
and 44% in L6-WT and Arg972-IRS-1 cells,
respectively, compared with the value in control L6 cells. Compared
with L6-WT cells, L6-Arg972 cells exhibited a
22% (P < 0.001) increase in basal GSK-3 activity
(Fig. 7). Insulin caused a significant
inactivation of GSK-3 in L6-WT, L6-Arg972, and
control L6 cells. Upon insulin stimulation, GSK-3 activity was
decreased by 49%, 32%, and 33% of basal values in L6-WT,
L6-Arg972, and control L6 cells, respectively.
However, inactivation of GSK-3 induced by insulin was reduced by 35%
in L6-Arg972 compared with L6-WT cells
(P < 0.001; Fig. 7).
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In the basal state, glucose incorporation into glycogen was 1.8-
and 1.36-fold higher in L6-WT and Arg972-IRS-1
cells, respectively, compared with that in control L6 cells (0.22, 0.4,
and 0.3 nmol/mg·min for control L6, L6-WT, and
L6-Arg972, respectively; P <
0.0001). However, in L6-Arg972 cells, basal
incorporation into glycogen was decreased by 24% compared with that in
L6-WT cells (P < 0.001). At submaximal insulin
concentrations, glucose incorporation into glycogen was significantly
increased in L6-WT compared with L6-Arg972 and
control L6 cells (P < 0.001), whereas no differences
were observed between L6-Arg972 cells and control
L6 cells (Fig. 8). In response to
10-9 mol/L insulin, glucose incorporation into
glycogen was increased by 40%, 23%, and 21% over basal in L6-WT,
L6-Arg972, and control L6 cells, respectively
(P < 0.001; Fig. 8). The maximally insulin-stimulated
glucose transport was significantly increased in both L6-WT and
L6-Arg972 cells compared with control L6 cells.
In response to 10-7 mol/L insulin, glucose
incorporation into glycogen was increased by 95%, 68%, and 52% over
basal in L6-WT, L6-Arg972, and control L6 cells,
respectively. Compared with L6-WT, L6-Arg972
cells exhibited a 43% decrease in submaximal and a 29% decrease in
maximal insulin-stimulated glucose transport (P <
0.0005). The sensitivity to insulin was also impaired in
L6-Arg972 ED50 occurring at
12 nmol/L compared with 1.8 nmol/L in L6-WT (P <
0.0001; Fig. 8, inset). Similarly, insulin-stimulated
glycogen synthase activity was reduced by 39% in
L6-Arg972 compared with L6-WT cells
(P < 0.001).
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Discussion |
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The role of Arg972-IRS-1 variant in
impairing glucose tolerance at the level of each individual major
insulin target tissue has not been investigated. In the present
investigation this issue has been addressed by studying the effect of
the Arg972-IRS-1 variant on insulin-stimulated
glucose transport and metabolism in L6 skeletal muscle cells, an
insulin-responsive cellular model widely used for studies of
insulin action (33, 41). We found that in L6 muscle cells transfected
with either wild-type IRS-1 or Arg972-IRS-1
variant, the amounts of IRS-1 protein expressed were similar, thus
indicating that the GlyArg972 substitution
does not affect the level of expression of IRS-1. Insulin leads to
tyrosine phosphorylation of IRS-1, which promotes association with SH2
domains of the p85 regulatory subunit of PI 3-kinase, leading to
stimulation of PI 3-kinase. We explored the functional consequences of
the Arg972-IRS-1 variant on these effects. We
found that expression of the Arg972-IRS-1 variant
in L6 cells did not affect the ability of insulin to stimulate IRS-1
phosphorylation. By contrast, expression of the
Arg972-IRS-1 variant in L6 cells resulted in a
significant decrease in IRS-1-associated PI 3-kinase activity as a
consequence of defective binding of the p85 subunit of PI 3-kinase to
IRS-1. These results are consistent with previous studies with
transfected 32D-IR cells expressing Arg972-IRS-1
variant (35, 36) as well as with our own data with RIN ß-cells (42),
but they are discordant with data obtained from COS-7 cells, where a
slight, but not significant, decrease in binding of the p85 subunit of
PI 3-kinase to Arg972-IRS-1 variant was observed
(40). This disparity may be due to several factors. First, as studies
with COS-7 cells were carried out using cells transiently cotransfected
with expression vectors for the human insulin receptor and wild-type
IRS-1 or Arg972-IRS-1 variant, it is possible
that variability in transfection efficiencies and levels of expression
of recombinant proteins among experiments could have masked the effects
of Arg972-IRS-1 variant on binding to PI
3-kinase. Second, transfection of COS-7 cells with a large excess of
recombinant human insulin receptor resulted in a maximal
phosphorylation of both wild-type IRS-1 and
Arg972-IRS-1 variant, which, in turn, promoted
maximal association with the p85 subunit of PI 3-kinase in the absence
of insulin. Under these conditions, insulin was not able to induce a
further increase in the association of both wild-type IRS-1 and
Arg972-IRS-1 variant with the p85 subunit of PI
3-kinase, thus making difficult detection of functional differences
between the IRS-1 molecules. Finally, of course, we cannot exclude that
differences in cell types may explain the disparity between the present
data and those observed with COS-7 cells.
Studies with animal models lacking IRS-1 and IRS-2
Previous studies using IRS-1 knockout mice or cells derived from these animals have suggested that IRS-2 is able to compensate for IRS-1 deficiency more effectively in liver and pancreatic ß-cells than in skeletal muscle or brown adipocytes (8, 9, 43, 44). IRS-2 knockout mice showed progressive alterations in glucose homeostasis culminating in overt diabetes (12). Indeed, skeletal muscle from IRS-2 knockout mice showed normal insulin-stimulated glucose uptake when isolated from animals with near-normal blood glucose levels, but not from animals with high glucose levels, thus suggesting that prolonged hyperglycemia due to hepatic insulin resistance and ß-cell failure, rather than the lack of IRS-2 in skeletal muscle, was the main mechanism for insulin resistance measured in vivo (45). IRS-1 knockout mice showed resistance to metabolic effects of insulin in skeletal muscle where IRS-2 only partially compensates for IRS-1 deficiency (43). According to this view, we found that expression of Arg972-IRS-1 variant did not affect endogenous IRS-2 content, insulin-stimulated tyrosine phosphorylation of endogenous IRS-2, or insulin-stimulated association of endogenous IRS-2 with PI 3-kinase. Furthermore, the extent of increases in phosphorylation of endogenous IRS-2 and its association with PI 3-kinase in response to insulin were lower in L6 skeletal muscle cells compared to that in other cell types, consistent with previous studies (43, 45). Taken together, these data suggest that IRS-1 plays a major role in mediating insulin-stimulated glucose transport in skeletal muscle and that, in skeletal muscle, IRS-2 may be unable to entirely compensate for defects in IRS-1 signaling, such as those caused by the Arg972-IRS-1 variant.
Functional impact of Arg972-IRS-1 variant on glucose transport and glucose transporters
We observed that both basal and insulin-stimulated glucose transports were increased in L6 skeletal muscle cells overexpressing wild-type IRS-1 compared to those in control L6 cells. Insulin sensitivity was also significantly increased, as deduced by the lower concentration of insulin required to achieve half-maximal stimulation. Overexpression of wild-type IRS-1 also increased the basal plasma membrane content of both GLUT1 and GLUT4 as well as the extent of GLUT1 and GLUT4 translocation in response to insulin. By contrast, in L6 cells expressing the Arg972-IRS-1 variant, both basal and insulin-stimulated glucose transports were decreased compared with those in L6 cells expressing wild-type IRS-1, and these changes were associated with a reduction in the sensitivity to insulin. Both basal plasma membrane content and the amount of GLUT1 and GLUT4 translocated to the plasma membrane in response to insulin were significantly decreased in L6 cells expressing the Arg972-IRS-1 variant compared with those in L6 cells expressing wild-type IRS-1. However, Arg972-IRS-1 expression did not affect total GLUT1 and GLUT4 protein content, thus ruling out the possibility that changes in expression of the two glucose transporters isoforms would account for the present data. In L6 cells expressing Arg972-IRS-1, the degree of impairment in basal glucose transport activity mirrored the amount of GLUT1 and GLUT4 in the plasma membrane in the basal state, whereas the impairment in insulin-stimulated glucose transport was comparable to that of GLUT4 translocation to the plasma membrane in response to insulin. These data suggest that the Arg972-IRS-1 variant may evoke differential effects on GLUT1 and GLUT4, impairing basal recycling of GLUT1 and GLUT4, on the one hand, and altering insulin-dependent translocation of GLUT4 to the plasma membrane, on the other.
Role of Akt and GSK-3 in metabolic insulin signaling downstream to PI 3-kinase
To further explore the mechanisms by which the Arg972-IRS-1 variant affects insulin signaling, we examined additional targets of insulin action that are thought to be downstream of PI 3-kinase. It has been shown that the Ser/Thr kinase Akt, a downstream effector of PI 3-kinase, is activated upon insulin stimulation (28). Akt is activated by a dual mechanism involving either the direct binding of PI 3-kinase phospholipid products to the Akt pleckstrin homology domain or the serine/threonine phosphorylation by one or more Akt kinases, which may themselves be stimulated by the phospholipid products of PI 3-kinase. Recently, evidence has been provided that activation of Akt acts as a key enzyme linking PI 3-kinase activation to multiple biological function of insulin, including glucose transport and glucose transporter translocation to the plasma membrane (31, 32, 33, 34). Consistent with these observations, we found that expression of the Arg972-IRS-1 variant in L6 cells resulted in a significant decrease in Akt phosphorylation and activity as a consequence of defective IRS-1-associated PI 3-kinase. These results suggest that an insulin-regulated pathway involving IRS-1/PI 3-kinase/Akt activation plays a significant physiological role in mediating glucose uptake and glucose transporter translocation. GSK-3 has been implicated in the regulation of glycogen synthesis through a mechanism involving its phosphorylation and inactivation by Akt (30, 32). Consistent with this idea, we found that overexpression of wild-type IRS-1 caused a significant inactivation of GSK-3 both under basal condition and in response to insulin. Expression of the Arg972-IRS-1 variant in L6 cells resulted in a significant decrease in GSK-3 inactivation compared with wild-type IRS-1. Of note, the degree of impairment in both basal and insulin-stimulated GSK-3 inactivation mirrored the observed defect in PI 3-kinase/Akt activation. Glucose incorporation into glycogen and glycogen synthase activities was also significantly impaired in L6 cells expressing the Arg972-IRS-1 variant, and these defects were associated with a reduction in insulin sensitivity. From these data, we conclude that the inactivation of GSK-3 via the PI 3-kinase/Akt pathway is probably important for regulating glycogen synthesis in skeletal muscle cells.
Conclusions
Skeletal muscle is the primary site of insulin-stimulated glucose disposal (25), and a large part of the glucose incorporated into muscle cells is stored as glycogen in response to insulin. Thus, glucose uptake and glycogen synthesis in skeletal muscle play a pivotal role in the maintenance of glucose tolerance. Although the Arg972-IRS-1 polymorphism can explain only a fraction of the total insulin resistance observed in vivo, a combination of this polymorphism with environmental factors may account for the phenotype of impaired glucose tolerance. According to this view, in obese nondiabetic subjects, the Arg972 polymorphism is associated with a 50% reduction in insulin sensitivity compared with that in obese subjects without polymorphism, thus suggesting that this polymorphism may induce greater insulin resistance than would occur with obesity on its own (15). Our report provides evidence that the stable expression in skeletal muscle cells of the naturally occurring Arg972-IRS-1 variant causes an impairment in the ability of insulin to activate the PI 3-kinase/Akt/GSK-3 signaling pathway, thus leading to defects in glucose transport, glucose transporter translocation, and glycogen synthesis. Taken together, these findings suggest that the polymorphism at codon 972 of the IRS-1 gene might contribute to the development of insulin resistance.
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Footnotes |
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Received September 27, 1999.
Revised January 5, 2000.
Accepted January 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by membrane targeting
promotes glucose and system A amino acid transport, protein synthesis,
and inactivation of glycogen synthase kinase 3 in L6 muscle cells. Diabetes. 47:1006–1013.[Abstract]
Arg amino acid polymorphism in IRS-1 impairs
insulin secretion in pancreatic beta cells. J Clin Invest. 104:357–364.[Medline]
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) or absence (
) of 100 nmol/L
insulin for 5 min at 37 C. The cells were lysed, equal amounts of
proteins were immunoprecipitated with anti-IRS-1 antibody, and PI
3-kinase activity was measured as phosphorylation of
phosphatidylinositol and quantified using a phosphorimager. Data
represent three independent experiments, each performed in duplicate,
and are expressed as a percentage of the control value (L6-WT). Basal
PI 3-kinase activity for control L6, 6 ± 0.2; L6-WT, 11.7 ±
1.1; L6-Arg972, 10 ± 1.8. Insulin-stimulated PI
3-kinase activity for control L6, 30 ± 0.4; L6-WT, 100 ±
2.8; L6-Arg972, 68 ± 3.5.
) myotubes were incubated with
2-deoxy-D-[14C]glucose in the presence or
absence of insulin, and glucose uptake was measured. The data are the
mean ± SD of three independent experiments performed
in triplicate. Results are expressed as the percent increase over basal
transport. Basal glucose transport for control L6, 0.9 nmol/mg·min;
L6-WT, 1.87 nmol/mg·min; L6-Arg972, 1.40 nmol/mg·min.
Insulin-stimulated glucose transport for control L6, 1.36
nmol/mg·min; L6-WT, 4.04 nmol/mg·min; L6-Arg972, 2.43
nmol/mg·min. Inset, Half-maximal insulin concentration
for the stimulation of 2-deoxy-D-glucose transport
(ED50) expressed as nanomoles per L. ED50 for
control L6, 3.1 ± 0.05; L6-WT, 2.4 ± 0.08;
L6-Arg972, 6.7 ± 0.04.
